Gemcitabine-Induced Activation of Checkpoint Signaling Pathways That Affect Tumor Cell Survival

Abstract

Two signaling pathways are activated by antineoplastic therapies that damage DNA and stall replication. In one pathway, double-strand breaks activate ataxia-telangiectasia mutated kinase (ATM) and checkpoint kinase 2 (Chk2), two protein kinases that regulate apoptosis, cell-cycle arrest, and DNA repair. In the second pathway, other types of DNA lesions and replication stress activate the Rad9-Hus1-Rad1 complex and the protein kinases ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (Chk1), leading to changes that block cell-cycle progression, stabilize stalled replication forks, and influence DNA repair. Gemcitabine and cytarabine are two highly active chemotherapeutic agents that disrupt DNA replication. Here, we examine the roles these pathways play in tumor cell survival after treatment with these agents. Cells lacking Rad9, Chk1, or ATR were more sensitive to gemcitabine and cytarabine, consistent with the fact that these agents stall replication forks, and this sensitization was independent of p53 status. Interestingly, ATM depletion sensitized cells to gemcitabine and ionizing radiation but not cytarabine. Together, these results demonstrate that 1) gemcitabine triggers both checkpoint signaling pathways, 2) both pathways contribute to cell survival after gemcitabine-induced replication stress, and 3) although gemcitabine and cytarabine both stall replication forks, ATM plays differential roles in cell survival after treatment with these agents.Gemcitabine (2Ј,2Ј-difluoro 2Ј-deoxycytidine), a pyrimidine-based antimetabolite, is currently licensed for the treatment of pancreatic cancer. Recent clinical studies have also demonstrated extensive activity of this agent against a variety of additional neoplasms, including carcinomas of the ovary, lung, and breast, acute leukemias, and refractory lymphomas (Carmichael, 1998;Nabhan et al., 2001). Because of its widespread use, there is considerable interest in understanding factors that affect sensitivity and resistance to this agent. Earlier studies demonstrated that gemcitabine is taken into cells on concentrative nucleoside transporter 1 and phosphorylated to gemcitabine 5Ј-monophosphate by deoxycytidine kinase (Plunkett et al., 1996). Subsequent addition of 5Ј-phosphates results in the formation of gemcitabine diphosphate and gemcitabine triphosphate, both of which contribute to the antiproliferative effects of gemcitabine (Plunkett et al., 1996). Gemcitabine diphosphate inhibits ribonucleotide reductase, thereby depleting deoxyribonucleotide levels. Gemcitabine triphosphate is a substrate for replicative DNA polymerases and causes chain termination one base pair beyond the site of incorporation.Because gemcitabine inhibits replication, this drug is predicted to activate the S-phase checkpoint, a series of reactions that inhibit DNA synthesis and enhance survival when cells experience replication stress. According to current understanding, the kinases ATR and Chk1 play critical roles in this checkpoint. When replicati...

AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies

“…Chk2 depletion in gemcitabine-treated cells had no effect on entry into mitosis, in contrast to the premature entry into mitosis observed with Chk1 depletion (17). Finally, siRNA knockdown of ATR, ATM, or Chk1 led to sensitization of gemcitabine-treated cells, whereas Chk2 depletion had no effect (16).…”

“…However, there is strong literature precedent indicating a major role for Chk1 versus Chk2 when both kinases were assessed simultaneously. For example, gemcitabine treatment activates both Chk1 and Chk2, but Chk1 to a greater extent as measured by phosphospecific antibodies (16,17). Additionally, both Chk1 and Chk2 can phosphorylate cdc25A, but in gemcitabine-treated cells only Chk1 siRNA knockdown resulted in stabilization of cdc25A.…”

“…5 Profiling of cell cycle proteins gave results that are consistent with mechanism of action and checkpoint abrogation. Gemcitabine treatment of cells leads to the phosphorylation and activation of Chk1 and Chk2 and predominantly S-phase arrest (16). Activated Chk1 then directly phosphorylates cdc25A leading to proteolytic degradation (11, 17 -22) and downstream to increased phosphorylation of cdk2 (Tyr 15 ).…”

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

“…Although originally thought of as a molecular sensor for double-strand breaks, the involvement of g-H2AX in recognizing other types of DNA damage and cellular stresses is becoming more evident (22, 33 -35). Whether H2AX is phosphorylated and forms nuclear foci at sites of nucleoside analogue -induced stalled replication forks has not been studied in detail (36).…”

Knockdown of Chk1 sensitizes human colon carcinoma HCT116 cells in a p53-dependent manner to lidamycin through abrogation of a G2/M checkpoint and induction of apoptosis

“…[10][11][12][13][14] However, the effect of Chk1 inhibition on DNA-damage agents is dependent on the cell line and cytotoxic agent. [15][16][17][18][19][20] It has been reported that inhibition of Chk1 hyperactivated ataxia telangectasia mutated gene (ATM), Rad 3 related gene (ATR), and caspase-2 after γ-radiation and trigger a caspase-2-dependent apoptotic program in human tumor cells. 21 Our previous studies have showed that LDM induced alterations of Chk1 and Chk2 in various cancer cells, 22,23 but the potential effects of cell cycle checkpoint kinases on LDM cytotoxicity remain elusive.…”